Method for monitoring and prognosis of disease course of gastrointestinal tumors

ABSTRACT

The present invention provides a method for diagnosis, monitoring and prognosticating the disease course of gastrointestinal tumors, comprising determining the level of a cyclin dependent kinase inhibitor (CK1) in a sample and diagnosing the disease and/or recurrence of the disease or prognosticating the disease course from the level of said CK1 in the examined tumor cells. Furthermore the present invention provides a method for tailoring a suitable therapy for gastrointestinal tumors. Another aspect of the present invention are test kits for research and diagnostic purposes.

This application is a National Stage of International ApplicationPCT/EP03/00968, filed Jan. 31, 2003, published Aug. 7, 2003 under PCTArticle 21(2) in English; which claims the priority of EP 02002454.3,filed Feb. 1, 2002.

FIELD OF THE INVENTION

The present invention provides a method for monitoring andprognosticating the disease course of gastrointestinal tumors,comprising determining the level of a cyclin dependent kinase inhibitor(CKI) in a sample and diagnosing the disease and/or recurrence of thedisease or prognosticating the disease course from the level of said CKIin the examined tumor cells.

BACKGROUND OF THE INVENTION

Gastric cancer is one of the most common cancers in humans. It has beenthe second most cause of cancer death in the world during the twentiethcentury. Although decreasing in numbers of incidence over the pastyears, gastric cancer is still one of the most frequent causes ofcancer-related deaths worldwide. Especially in Asia the prevalence ofgastric cancer remains higher than in the western world.

Generally the prognosis for gastric cancer is still poor. This is inpart due to the fact, that the disease is often diagnosed in a latestage. Furthermore there is a high rate of recurrence after initialtherapy of the carcinoma. Therefore an important step in managinggastric cancer is diligent monitoring of the disease course.

First this means to determine the stage of the patients disease. Thedetermination of the stage has a potential prognostic value and can beused to design an optimal therapy. Although in gastric cancerpathological staging is generally preferable over clinical staging, theadvantage of clinical staging is, that it does not depend on surgicallyinvasive methods. Thus characterization of the molecular biologicalproperties of a particular tumor could lead to a more specific andefficient therapy. According to the molecular basics of the tumor atherapy could be tailored to avoid recurrence of the disease.

Furthermore monitoring means a close follow up of the disease afterinitial therapy. On the basis of classical clinical methods thedetection of the recurrence of tumors is quite insensitive, so that thedisease has reached a progressed stage until it is found. The follow upcould as well be carried out on a molecular level, to recognize therecurrence of the disease as early as possible.

There is a series of tools to assess primary diagnosis in tumors such asgastrointestinal tumors. Yet, due to the diversity of the molecularcharacteristics of tumors, the outcome of detected tumor may varywidely. For assessing prognosis and tailoring an adequate therapyfurther characterization of the tumors is indispensable. In a series oftumors prediction about the course and the treatment necessary can bemade by testing for the level of expression of several tumor markers.Based upon this prognosis it is possible to choose a treatment for theparticular tumor to ensure the best chances for the patient gatheredwith lowest necessary therapeutical burden. For gastric cancer theclassic ways of staging and grading of the tumor allow only for arestricted prognosis, so that actually consuming therapies are putthrough to avoid recurrence of tumors. If the aggressiveness of tumorscould be diagnosed on the basis of molecular markers, the therapy couldbe better suited to the needs of the special case.

Some marker proteins for diagnosing of gastric cancer have beenidentified to date. For a reliable prognosis of the disease course ofgastric cancer in individual patients a series of tumor related cellcycle regulatory proteins have been tested. Yet no correlation betweentumor markers and the prognosis of disease course could be found up tonow.

Molecular markers being useful for the prognosis of the disease coursein a wide range of tumors are the inhibitors of cyclin-dependentkinases. The key role of cyclin dependent kinase inhibitors in the cellcycle is the regulation of the activity of the cyclin dependent kinases.This regulation is brought forth by binding of the CKI to specificbinding sites on their respective binding partner cyclin dependentkinases. Two main families of CKI have been identified, the members ofwhich share a high percentage of sequence homology and the bindingspecificity to their binding partners. The first family ofcyclin-dependent kinase inhibitors binds specifically to and inhibitscdk2. The second family in contrast preferentially binds to and inhibitscdk4 and cdk6. Members of the second family of CKI are for example p16,p15, p18 and p19/20.

One candidate marker for the prognosis of disease course, that hasproven useful in several tumor types, is the MTS1 protein (p16^(INK4A)).p16 has been reported to be valuable marker for assessment of thebiological behavior and prognosis for example in nasopharyngealcarcinoma (Wang, L. et al.; 1999, 30 (4), 394-396) and breast cancer. Inthese cases loss of p16 expression indicates poor prognosis forpatients.

Another cyclin-dependent kinase inhibitor p27 turned out to be acandidate for a prognostic marker in tumors.

The level of expression of p27 protein has been described to allowassessment of prognosis in a wide range of tumors (US6180333). In thecase of p27, expression of the protein within the samples is associatedwith better prognosis. The cumulative survival of patients that did notexpress p27 in the tumor tissue is significantly reduced compared to thepatients that showed p27 expression. In gastrointestinal tumors thereare hints for correlations between tumor progression and reduced levelsof p27 protein (Migaldi M., et al., Pathol Res Pract 2001;197(4):231-6),yet there are difficulties in assessing reliable prognosis ingastrointestinal tumors using this marker protein (Feakins R M., et al.,Cancer 2000 Oct. 15;89(8): 1684-91).

Especially in gastrointestinal cancers there is need for molecularmarkers, that allow for assessment of prognosis, diligent monitoring,building a strategy for therapy and finally sensitive followup.

This is provided by the method claimed according to the presentinvention.

SUMMARY OF THE INVENTION

The present invention is based on the inventors findings shown in theexamples 1-3, that the level of expression of cyclin-dependent kinaseinhibitors in tumor samples allows to diagnose and grade the malignancyof a particular tumor, to predict the course of the disease and tofollow up the disease after initial therapy. Especially the inventorsfound that the overexpression CKI is correlated with poor prognosis.Overexpression as used herein shall mean an expression at least two foldelevated in comparison to wild type levels. The present inventionfurthermore provides a method, that allows to build a strategy for thetherapy of tumors according to their molecular properties. According tothe present invention the level of cyclin-dependent kinase inhibitors(CKI) can be used as a molecular marker for assessing prognosis,monitoring and the design of a strategy of tumor therapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Kaplan-Meier plots correlated to the data ofimmunohistochemical determination of p16 protein expression in tumorresection samples of node negative tumors of the stomach. On the x-axisof the plot the survival of the individuals is given in months. The yaxis gives the cumulative survival. The shown curves are named positive,which means individuals overexpressing p16 protein in the tumor samples,and negative, which means individuals not overexpressing p16 protein inthe tumor resections. For the negative samples the cumulative survivalof the tested individuals is reduced by about 20% over the tested periodof 160 months. In the case of the p16 overexpressing samples thecumulative survival of all tested individuals in contrast is reduced bymore than 70% over the same period of time.

FIG. 2 shows the clinical and pathological characterization of allpatients included in a study on the prognostic value of differentmolecular markers with respect to gastric cancer. Shown are the resultsof the univariate Kaplan-Meier analysis (p-values) for all patients,nodal-negative cases only (pN0) as well as nodal-positive cases only(pN1+). For experimental details see Example 4.

FIG. 3 show the multivariate analysis (Cox-model) of the clinicalpatient data and results of immunohistochemical staining ofgastrectomies; the Table includes data concerning the factors:localization, age, gender, pT-stage, pN-stage (excluded for subanalysisof nodal-negative and nodal-positive cases), histology, grading, p16.Only the factors which showed a significant prognostic value forsurvival are listed in this table. Further factors, that have beeninvestigated during the studies are e.g. p53, Ki-67, CK18, Her2, S100A4,Cyclin D1, Cyclin D3 and p27. Abbreviations: RR: relative risk, CI: 95%confidence intervall for relative risk. For experimental details seeExample 4.

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis as used in the context of the present invention may comprisedetermining the level of CKI in a sample. Based upon the determinedlevel of CKI in the samples individuals can be subdivided intosubgroups. The subgroups may be created according to clinical data, suchas e.g. survival, recurrence of disease, frequency of metastases etc.,related to the particular level of CKI determined in the samples.

Based upon these subgroups an assessment of prognosis may be done.According to the subgroups the therapy of the individuals affected bythe tumors may be tailored.

Monitoring may comprise detecting the level of CKI in samples taken atdifferent points in time and determining the changes in said level.According to said changes the course of the disease can be followed. Thecourse of the disease may be used to select therapy strategies for theparticular individual.

Another aspect of diagnosis and monitoring of the disease courseaccording to the present invention may comprise the detection of minimalresidual disease. This may comprise for example the detection of a CKIlevel in one or more body samples following initial therapy of anindividual once or at several timepoints. According to the level of CKIdetected in the samples one may select a suitable therapy for theparticular individual.

Gastrointestinal tumors as used in the context of the present inventionare all tumors of the gastrointestinal tract. Tumors may comprise forexample neoplasms such as benign and malignant tumors, carcinomas ordysplasias. In a preferred embodiment of the present invention thegastrointestinal tumor is for example a tumor of the esophagus, thestomach, the pancreas, the bile tree, the liver, the small intestine,the colon or the rectum. In a more preferred embodiment the tumor is forexample cancer of the esophagus, gastric cancer, cancer of thegallbladder, the pancreas, the liver, the small intestine, the colon orthe rectum. The tumors according to the present invention may comprisetumors, which show detectable lymph-node involvement (node positivetumors) as well as tumors, without detectable spread to lymphnodes (nodenegative tumors). In one preferred embodiment of the invention thegastrointestinal tumors are tumors without detectable spread to lymphnodes.

A sample according to the method of the present invention is any sampleof cells or body fluids containing cell cycle regulatory proteins. Suchsamples may be for example gastrointestinal secretions, stool, bile,biopsies, cell- and tissue-samples. Biopsies as used in the context ofthe present invention may comprise e.g. resection samples of tumors,tissue samples prepared by endoscopic means or needle biopsies oforgans. Furthermore any sample potentially containing the markermolecules to be detected may be a sample according to the presentinvention. Such samples may comprise for example intact cells, lysedcells or any liquids containing proteins, peptides or nucleic acids.Even solids, to which cells, cell fragments or marker molecules, such asCKI nucleis acids or CKI proteins, may adhere, may be samples accordingto the present invention. Such solids may comprise for examplemembranes, glass slides, beads etc.

Preparation of a sample may comprise e.g. obtaining a sample of atissue, a body fluid, of cells, of cell debris from a patient. Accordingto the present invention preparation of the sample may also compriseseveral steps of further preparations of the sample, such as preparationof dissections, preparation of lysed cells, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids or preparation of beads, membranesor slides to which the molecules to be determined are coupled covalentlyor non-covalently.

The cyclin-dependent kinase inhibitors according to the presentinvention may for example be CKI, that do not preferentially bind tocdk2. So CKI according to the present invention may be for example theCKI of the second family. The CKIs used for the method according to thepresent invention bind preferentially to cdk4 or cdk6 and have low or nobinding specificity for cdk2. In one preferred embodiment the CKI may bep16. The present invention may also be applicable to fragments of CKI,functionally equivalent sequences of CKI proteins or nucleic acidsequences coding for functionally equivalent CKI proteins and sequencesof CKI proteins or coding for CKI proteins, that are mutated or alteredin any way. The mutations according to the present invention maycomprise for example insertions, deletions, substitutions or nucleotidemutations.

The method for detection of the level of the cyclin-dependent kinaseinhibitor according to the present invention comprises any method, whichis suited to detect very small amounts of specific biologically activemolecules in biological samples. The detection reaction according to thepresent invention may be for example a detection either on the level ofnucleic acids or on the level of polypeptides.

In one preferred embodiment of the invention the detection of the levelof CKI may be carried out by detection of the level of nucleic acidscoding for the CKI or fragments thereof present in the sample. The meansfor detection of nucleic acid molecules are known to those skilled inthe art. The procedure for the detection of nucleic acids may forexample be carried out by a binding reaction of the molecule to bedetected to complementary nucleic acid probes, proteins with bindingspecificity for the nucleic acids or any other agents specificallyrecognizing and binding to said nucleic acids. This method may beperformed as well in vitro as directly in situ for example in the courseof a detecting staining reaction. Another way of detecting the CKI in asample on the level of nucleic acids performed in the method accordingto the present invention may be an amplification reaction of nucleicacids, which may be carried out in a quantitative manner such as forexample the polymerase chain reaction. In a preferred embodiment of thepresent invention real time RT PCR may be used to quantify the level ofp16 RNA in samples of tumors.

In another preferred embodiment of the invention the detection of thelevel of CKI may be carried out by determining the level of expressionof a protein. The determination of the CKI on the protein level may forexample be carried out in a reaction comprising antibodies specific forthe detection of the cyclin-dependent kinase inhibitor. The antibodiesmay be used in many different detection techniques for example inwestern-blot, ELISA or immuno-precipitation. Generally antibody baseddetection may be carried out as well in vitro as directly in situ forexample in the course of an immuno-histochemical staining reaction. Anyother method for determining the amount of particular polypeptides inbiological samples may be used according to the present invention.

In a preferred embodiment of the invention the level of CKI issignificantly, e.g. at least 2-fold, elevated compared to a non tumoroustest sample. In this case the CKI is overexpressed in the sample.

Another aspect of the present invention is a testing kit for performingthe method according to the present invention. The kit may be forexample a diagnostic kit or a research kit.

A kit according to present invention may comprise:

a) reagents for the detection of the cyclin-dependent kinase inhibitor

b) the reagents and buffers commonly used for carrying out the detectionreaction, such as buffers, detection-markers, carrier substances andothers

c) a cyclin-dependent kinase inhibitor sample for carrying out apositive control reaction

The reagent for the detection of the cyclin-dependent kinase inhibitormay include any agent capable of binding to the cyclin-dependent kinaseinhibitor molecule. Such reagents may include proteins, polypeptides,nucleic acids, peptide nucleic acids, glycoproteins, proteoglycans,polysaccharids or lipids.

The cyclin-dependent kinase inhibitor sample for carrying out a positivecontrol may comprise for example nucleic acids in applicable form, suchas solution or salt, peptides in applicable form, tissue section samplesor positive cells.

In a preferred embodiment of the invention the detection of thecyclin-dependent kinase inhibitor is carried out on the level ofpolypeptides. In this embodiment the binding agent may be for example anantibody specific for the cyclin-dependent kinase inhibitor or afragment thereof.

In an other embodiment of the test kit the detection of the cyclindependent kinase inhibitor is carried out on the nucleic acid level. Inthis embodiment of the invention the reagent for the detection may befor example a nucleic acid probe or a primer reverse-complementary tosaid cyclin-dependent kinase inhibitor nucleic acid.

The method according to the present invention may be used to furthercharacterize detected tumors of the gastrointestinal tract. The methodalso allows to prognosticate the course of the disease, to design anadequate therapy for the particular tumor and to monitor the diseasecourse in all stages from the primary diagnosis throughout the initialtherapy to the follow up. Especially the method according to theinvention provides means for staging of tumors without depending onsurgically invasive methods. The method according to the inventionprovides simple to interpret results even in cases, where the classicalcytological and histological methods rely on subjective opinions. Oneadvantage of the method is its simplicity in handling, which makes itapt for routine diagnosis and screening tests. Thus the method accordingto the present invention provides a tool that enhances the diagnosis andmonitoring and that allows for assessment of the prognosis of therespective tumor patient. The present invention furthermore provides atool for subdividing patients into particular subgroups with respect tothe prognosis based upon the level of CKI. According to these subgroupsof CKI levels the therapy of the individuals may be tailored.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

EXAMPLES Example 1 Determining Correlations Between the Level ofExpression of p16 in Stomach Carcinoma Biopsies and the Survival Rate ofthe Patients

In order to determine, whether there is a correlation between thepatients outcome and the level of expression of p16 protein detectablein tumors, preparations of 373 gastrectomies were analysed.

The immuno-histochemical detection reaction is carried out as follows(washing steps are included following each single incubation reaction):The paraffin sections are deparaffinized in xylene for 2×10 min. andrehydrogenated using descending dilutions of ethanol (100%, 100%, 90%,80%, 70%, 50%). The antigens are demasked in 10 mM citrate buffer (pH6,0) in a waterbath at 95° C. for 40 min. Thereafter the endogeneousperoxidases are inactivated using 3% H₂O₂ in PBS. Then the blocking ofunspecific binding sites with 1% Casein is carried out at roomtemperature for 20 minutes. Thereafter the sections are incubated with amonoclonal antibody binding p16 at room temperature for 60 minutes. Forthe detection of the p16-antibody binding a biotin labelled bioRAM(rabbit-anti-mouse) antibody is added for 30 min. at 37° C. and thenavidin-biotinylated peroxidase is added for 30 min. at room temperature.Following, biotinylated thyramid with 0,003% H₂O₂ is added (DAKOCSA-Kit). Thereafter avidin-biotinylated alkaline phosphatase is addedfor 30 min. Then the chromogen neo-fuchsin (pH 8,7) is added for 30 min.A nuclear counterstain is carried out using hemalaun solution.

Alternatively the immuno-histochemical detection reaction is carried outas follows: The paraffin sections are deparaffinized in xylene for 2×10min. and rehydrogenated using descending dilutions of ethanol (100%,100%, 90%, 80%, 70%, 50%). The antigens are demasked in 10 mM citratebuffer (pH 6,0) in a waterbath at 95° C. for 40 min. Thereafter theendogeneous peroxidases are inactivated using 3% H₂O₂ in PBS. Followingthe blocking of unspecific binding sites with horse serum at roomtemperature for 20 minutes, the sections are incubated with ap16-specific monoclonal antibody (Neomarkers, Fremont, Calif., U.S.A.)in the presence of 3% fetal calf serum at room temperature for 45minutes. For the detection of the p16-antibody binding EnVision System(DAKO A/S, Glostrup, Danmark) comprising anti mouse IgG coupled to aHRP-labeled dextran polymer is added for 30 min. DAB is used thereafteras substrate for the detection reaction and a nuclear counterstain iscarried out using Mayer's hemalaun solution.

Following the immuno-histochemical analysis of the tissues the resultswere compared to the correlating clinical data. Especially Kaplan-Meierplots were done referring to clinical data corresponding to theparticular preparations of the tumor resections in correlation to thelevel of expression of p16 protein found in the samples. The KaplanMeier plots for the resection samples of node negative tumors are shownin FIG. 1. The plots show, that the total survival of individuals, whoseresection samples showed p16 overexpression, is clearly reduced comparedto the survival of individuals, which do not show overexpression of p16in the samples. As a parameter for determination of positive or negativesamples the percentage of stained cells were chosen. Samples with morethan 33% of the cells in an area stained are said to be overexpressingp16 and thus to be positive. Negative samples not overexpressing p16show less than 33% of stained cells in the tested area of the samples.For the negative samples, not showing p16 overexpression, the cumulativesurvival of the tested individuals is reduced by about 20% over thetested period of 160 months. In the case of the p16 overexpressingsamples the cumulative survival of all tested individuals in contrast isreduced by more than 70% over the same period of time.

Furthermore the investigations show, that nodal negative cases ofcarcinoma of the stomach not showing overexpression of p16 protein inthe immuno-histochemical analysis have a significantly higher survivalrate than those cases showing p16 protein overexpression. Thisillustrates the correlation between survival of the affected individualsand the level of overexpression of p16 protein in tumor tissues. Theexample shows that the method according to the present inventionprovides a means for prognosis of the outcome of patients based on thedetection of the level of expression of p16 protein in tumor biopsies.

Example 2 Determining the Correlation Between Survival Rate and p16 mRNALevels in Tumor Tissues

Dissections of tumor biopsies can be semi-quantitatively analysed forthe mRNA level of p16 in a in-situ staining reaction. The stainingreaction is performed as follows:

The tissue dissections are incubated in ascending ethanol concentrationsup to 100% ethanol. After evaporation of the alcohol the dissections areboiled in 10 mM citrate buffer (pH 6,0) for pre-treatment of the tissue.The hybridization mixture is prepared by mixing 50 μl of ready to usehybridisation buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 pmolof the probes. The probes are fluorescein-labelled oligonucleotides ofthe following sequence:

ccttt taacg tagata taagc cttcc c (SEQ ID NO: 1)

The hybridisation mixture is heated to 95° C. and afterwardsequilibrated to 37° C. After the boiling procedure the dissections areincubated with each 50 μl of the hybridisation mixture for 2 hours at37° C. The dissections are washed in excess volumes of the wash bufferstwo times in 2×SSC at room temperature for 15 min. and once in 1×SSC at50° C. for 15 min. Then the dissections are rinsed two times at roomtemperature in 2×SSC. Following this washing procedure the dissectionsare incubated for 30 min. with blocking buffer (NEN, Blockingpugger) atroom temperature. Then follows 1 hour incubation with a 1:100 diluted(in Blocking buffer, see above) Anti-Fluorescein-AP (DAKO A/S). Thedissections are then washed 2 times in 1×PBS/0, 1% Tritonx100 for 10min. at room temperature, followed by one wash step with 1×PBS, 50 mMMgCl₂ (pH 9,2) for 10 min. at room temperature. Then the stainingreaction is performed with NBT/BCIP (Sigma) for about 30 min. at roomtemperature. The staining reaction is stopped by a short incubation with1 mM EDTA in PBS. Finally the dissections are dipped in H₂O_(dest) andfixed with AquaTex (Merck). Then the stained dissections can be analysedmicroscopically.

When the histochemical data are compared to clinical data as describedin Example 1, the level of p16 RNA detectable in the in situ reactioncorresponds to the survival rate of the individuals in a similar manneras shown in Example 1.

Example 3 Determination of Correlation Between Survival Rate and p16Level in Tumor Tissues using Semiquantitative RT PCR

Samples of liver carcinomas are used to determine the level of p16 mRNAusing semi-quantitative RT PCR. 34 tumor biopsies are used in thisstudy.

Tumors are collected, snap frozen, and stored at −80° C. They areverified to be composed predominantly of neoplastic cells byhistopathological analysis. mRNA is isolated from tumors andpatient-matched normal liver tissue using Qiagen reagents (Qiagen,Hilden, Germany), and single-stranded cDNA is synthesized usingSuperscript II (Life Technologies, Inc.). Quantitative PCR is performedusing the 7700 Sequence Detector (Taqman™) and the SYBR Green PCRMaster-Mix, as described in the manufacturers manual (AppliedBiosystems, Foster City, Calif.).

PCR reactions are performed in 25 μl volumes with a final concentrationof 300 nmol for each primer, with 95° C. for 15 sec and 60° C. for 60sec, for 40 cycles. The following primers are used for quantitative PCR:

p16-A: TCACT GTGTT GGAGT TTTCT GG (SEQ ID NO: 2) and p16-B: GCTTC CCTAGTTCAC AAAAT GC (SEQ ID NO: 3)

The specificity of the PCR products is verified by gel electrophoresis(data not shown).

The data concerning to the level of p16 expression are correlated toclinical data related to the particular tumor samples. The comparison ofthe p16 levels in the tissues correlates to the outcome of the patientsin a similar manner as shown in Example 1.

This illustrates, that the method according to present inventionprovides a means for determining parameters strictly correlated to theclinical data such as survival and course of the disease on a molecularlevel. The method may be used according to the present invention toassess prognosis of patients on the basis of molecular biological data.

Example 4 Determination of the Prognostic Value of Different MolecularMarkers with Respect to Gastric Cancer

The present example makes use of tissue micro arrays for investigationof putative prognostic markers in the course of a comprehensiveimmunohistochemical analysis of characterized gastric carcinomas.

373 cases with primary gastric cancers fulfilling the followingcriteria: a) adequate paraffin blocks available, b) UICC-R0 resection,c) pM0 status, d) histologic confirmation of gastric carcinoma e) lackof a history of radiation or chemotherapy, previous gastric resection orsecond malignancy, e) follow-up data available, f) no perioperativelethality (survival longer than two month after surgery) are examined inthe present experiment. All patients underwent gastrectomy with lymphnode dissection.

Tissue micro arrays (TMA) for use in the experiments are assembled asfollows: all diagnostic H&E slides from each case are reviewed andrepresentative and well preserved tumor tissue is circled on theappropriate slides with a permanent marker. This area of interest istransferred to the cutting side of the corresponding paraffin block andalso defined with a permanent marker. Care is taken to choose an areathat has been fixed properly and does not show necrosis or scarredtissue. Using a sharpened needle as punching device, tumor tissue coresare taken from each case and stored in a microfuge tube for later use.Finally, the tissue cores of sixty cases are melted together into onenew homogenous multiblock that had properties identical to those of anystandard paraffin block. Overall, eight tissue micro arrays are preparedcontaining all study cases. TMA are cut and slides were mounted in thesame way as with any conventional paraffin block.

Staining is performed on 2 μm sections of the multiblocks. Slides aredewaxed by xylene, rehydrated by graded alcohol and epitope retrieval iscarried out by heating the slides for 20 minutes (100° C.) in 10 mMsodium citrate (pH 6.0). Sections are stained using the Shandoncoverplate system in a Tecan Genesis Autostainer (Shandon, Frankfurta.M., Germany; Tecan, Deisenhofen, Germany). Tissue peroxidase activityis blocked by incubation with 3% hydrogen peroxide for 8 minutes. Theprimary p27 antibody (novocastra, UK) is applied in a dilution of 1:125.Further molecular markers that are investigated during the studies aree.g. p53, Ki-67, CK18, Her2, S100A4, Cyclin D1 and Cyclin D3.

Biotinylated secondary antibodies are used for the catalyzed signalamplification technique (CSA, Dako). The final color reaction is carriedout using new fuchsin as a chromogen, hemalaun as light counterstainingand Kaiser's glycerine (Merck, Darmstadt, Germany) to mount thecoverslips.

For the markers six groups are evaluated on a percentual basis: 0-1%;−5%; −10%; −33%; −50% and over 50% of tumor cells showing specificpositivity. A preliminary analysis is performed with regard to clinicaloutcome. On the basis of this preliminary statistical analysis, groupsthat showed an identical or similar (p>0.2) course are combined. Tosimplify further evaluation, a bimodal distribution is finallycalculated for each parameter including a high-risk and a low-riskgroup. All further analysis is based on this bimodal distribution.

To investigate associations between expression of the markers andseveral clinico-pathological and other cell-cycle parameters, data arecross-tabulated and Fisher's exact test is performed (see FIG. 2). Theassociation of staining for p16 with patient survival is evaluated usinglife tables constructed from survival data with Kaplan-Meierplots,—comparisons between groups are performed with log-rank test. TheCox proportional hazards model (multivariate analysis) is applied toassess the predictive value of the markers, using both a forward and abackward stepwise selection of significant factors (p<0.05). Tests arecarried out separately for all cases, or for nodal-negative andnodal-positive status only. All statistical analyses are performed usingSPSS version 10.0.7 (SPSS Inc., IL/USA).

For CKI p27 a higher protein expression is associated with a worseprognosis in our series parallel to p16 results. In the present study,p27 does not prove its prognostic value in multivariate regressionanalysis if CKI p16 is added to the model. Thus, p16 overexpressionremains the only immunohistochemical marker protein that providesprognostic information in all cases in addition to localization, age,tumor stage (pT) and nodal stage (pN). Even more strikingly, in astratified model focusing on nodal-negative patients, p16 overexpressionremains the only factor with prognostic value in multivariate analysis(compare FIG. 3).

Thus the results of the present example show that the overexpression ofp16^(INK4A) is a powerful prognostic marker molecule. The experimentsrevealed, that the findings regarding the prognostic value of a seriesof molecular marker molecules may not be verified analyzing acomprehensive panel of gastric carcinoma samples employing the describedhigh throughput methods.

The cyclin dependent kinase inhibitor p27 interacting with cdk2 that hasbeen included in the present study could not prove any prognostic value.This finding indicates that cyclin dependent kinase inhibitors that donot preferentially bind to cdk4 or cdk6 but e.g. to cdk2 are not assuitable for use in the presented method as CKI preferentially bindingto cdk4 or cdk6.

It could be stated that prognostic value for cyclin dependent kinaseinhibitors could only be shown for the CKI binding preferentially tocdk4 or cdk6. For these cyclin dependent kinase inhibitorsoverexpression could be shown to be associated with poor prognosis ingastric carcinomas.

Example 5 Determination of Correlation Between Survival Rate and p19Level in Carcinomas of the Small Intestine using Semiquantitative RT PCR

Samples of carcinomas of the small intestine are used to determine thelevel of p19 mRNA using semi-quantitative RT PCR. 41 tumor biopsies areused in this study.

Tumors are collected, snap frozen, and stored at −80° C. They areverified to be composed predominantly of neoplastic cells byhistopathological analysis. mRNA is isolated from tumors andpatient-matched normal intestinal mucosa using Qiagen reagents (Qiagen,Hilden, Germany), and single-stranded cDNA is synthesized usingSuperscript II (Life Technologies, Inc.). Quantitative PCR is performedusing the 7700 Sequence Detector (Taqman™) and the SYBR Green PCRMaster-Mix, as described in the manufacturers manual (AppliedBiosystems, Foster City, Calif.).

PCR reactions are performed in 25 μl volumes with a final concentrationof 300 mmol for each primer, with 95° C. for 15 sec and 60° C. for 60sec, for 40 cycles.

The specificity of the PCR products is verified by gel electrophoresis(data not shown).

The data concerning the level of p19 expression are correlated toclinical data related to the particular tumor samples. The results show,that the outcome of individuals bearing carcinomas of the smallintestine, that overexpress p19 mRNA, is poor in comparison to theoutcome of those bearing carcinomas not overexpressing p19 mRNA.

This illustrates, that the method according to present inventionprovides a means for determining parameters strictly correlated to theclinical data such as survival and course of the disease on a molecularlevel. The method may be used according to the present invention toassess prognosis of patients on the basis of molecular biological data.

1. A method for prognosticating the survival rate of an individualhaving lymph node-negative gastric cancer comprising detecting theprotein expression level of cyclin-dependent kinase inhibitor p16 in agastric cancer biopsy sample obtained from the individual, wherein theoverexpression of the cyclin-dependent kinase inhibitor p16 iscorrelated with the prognosis of reduced survival rate of theindividual.
 2. The method of claim 1, wherein the sample is coupledcovalently or non-covalently or adhere to a solid.
 3. The method ofclaim 1, wherein the detection of the protein expression level iscarried out by means of an antibody specific for the cyclin-dependentkinase inhibitor p16.